Class II major histocompatibility complex (MHC) gene products play critical roles in a variety of T and B lymphocyte responses. Biochemical and functional analyses have been used to investigate the relationship between class II structure and peptide antigen presentation. Crystallographic analysis of MHC class II molecules suggested that they could self-associate into "dimers of dimers" and that this might affect T cell activation. We have used mutagenesis, transfection, and in vitro T cell assays to test this model, and have identified sets of alpha and beta chain residues lying in the putative dimerization interface that when altered to alanines decrease T cell activation without detectable changes in either peptide binding or gross MHC molecule conformation. If tests with soluble T cell receptors confirm a lack of effect on T cell recognition per se, these data will provide strong evidence in favor of a role for class II self-association during T cell antigen recognition in effective signaling by the TCR. In addition to binding peptide and being recognized by clonally distributed T cell receptors, class II molecules participate in T cell selection in the thymus and mature T cell activation in the periphery by interacting with the CD4 molecule that is also the primary receptor for HIV-1. We have previously used site-directed mutagenesis and in vitro functional assays to define the site(s) of interaction of class II molecules with CD4. We have now gone on to show that short peptides consisting of the linear sequence from the primary site on MHC class II molecules controlling function with CD4 can affect CD4-dependent T cell responses in vitro and in vivo. These effects can be either positive or negative, depending on the antigen stimulation conditions used. We have also constructed transgenic mice whose expressed class II molecules all contain a mutant beta chain defective in interaction with the CD4 coreceptor. These mice are deficient in selecting CD4+ T cells for maturation in the thymus, confirming that the site we identified as important for CD4-class II interactions based on responses of immortalized cells in vitro is also involved in physiologic class II-CD4 interactions in vivo. The antigen-presenting cells from these mice will constitute a useful source of non-transformed cells for tests of the effects on T cell responses and tolerance induction of defective CD4 recruitment during primary and secondary exposures of T cells to MHC class II-peptide ligands.